title: Optimization of rapid Salmonella enterica detection in liquid whole eggs by SYBR green I-based real-time reverse transcriptase-polymerase chain reaction.
abstract: Eggs and egg products have a high risk of Salmonella enterica serovar Enteritidis contamination leading to gastroenteritis outbreaks in humans. Thus, a rapid screening tool for viable Salmonella Enteritidis cells in the egg industry is needed. Our objective was to rapidly and sensitively detect viable Salmonella Enteritidis from spiked liquid whole eggs (LWEs) within 24 h using SYBR green I-based real-time reverse transcriptase-polymerase chain reaction (PCR) targeting the Salmonella specific invA gene along with an internal amplification control in a Bio-Rad iCycler. LWE was inoculated with Salmonella Enteritidis and mixed with tetrathionate broth, and 100 μL of serially diluted portions in phosphate-buffered saline was plated on Xylose Lysine Tergitol 4 agar or 5 mL were used for RNA extraction by the TRIzol method immediately or after enrichment of 6, 12, or 16 h at 37 °C. The real-time reverse transcriptase-PCR assay was carried out using previously described Salmonella invA gene primers. Melt temperature analysis of the PCR product was included to determine specific invA amplification. Without enrichment, the assay detection limit was 10(7) colony forming units (CFU)/25 mL LWE. After enrichment for 6 and 12 h, Salmonella Enteritidis could be detected from LWE up to 10(4) and 10(2) CFU/25 mL, respectively. Improved Salmonella Enteritidis detection up to 10(0) CFU/25 mL was obtained after 16-h enrichment. Even with 16-h enrichment, the results could be still be obtained within 24 h, which is much faster than by traditional cultural detection that takes several days. Therefore, this assay appears suitable for routine detection of Salmonella enterica contamination by the egg industry to help prevent the transmission of egg-associated Salmonella outbreaks and timely recall of contaminated products.
authors.affiliation: Department of Food Science and Technology, University of Tennessee, Knoxville, Tennessee 37996-4591, USA.
authors.forename: Chayapa
authors.initials: C
authors.lastname: Techathuvanan
authors.forename: Doris Helen
authors.initials: DH
authors.lastname: D'Souza
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics; D052160:Benzothiazoles; D015169:Colony Count, Microbial; D003959:Diamines; D004531:Eggs / Q000382:microbiology*; D005456:Fluorescent Dyes / Q000737:chemistry; D005513:Food Inspection / Q000379:methods*; D005516:Food Microbiology; D005798:Genes, Bacterial; D057230:Limit of Detection; D050296:Microbial Viability; D058889:Molecular Typing; D009930:Organic Chemicals / Q000737:chemistry; D016133:Polymerase Chain Reaction; D011804:Quinolines; D012329:RNA, Bacterial / Q000302:isolation & purification / Q000378:metabolism; D012333:RNA, Messenger / Q000737:chemistry / Q000378:metabolism; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction; D019779:Salmonella enterica / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*; D012477:Salmonella enteritidis / Q000145:classification / Q000235:genetics / Q000302:isolation & purification; D013696:Temperature; D013997:Time Factors
chemical_list: D001426:Bacterial Proteins; D052160:Benzothiazoles; D003959:Diamines; D005456:Fluorescent Dyes; D009930:Organic Chemicals; D011804:Quinolines; D012329:RNA, Bacterial; D012333:RNA, Messenger; C075518:invA protein, Bacteria; C098022:SYBR Green I